Streptococcus mutans, the cause of dental caries, contains antigens crossreactive with human cardiac and skeletal muscle. This finding requires caution in development of anti-caries vaccines and demands intensive investigations of the immunochemistry of walls and membranes of this oral inhabitant. Recent studies of the S. mutans BHT membrane have revealed it to be the site of heart cross-reactive antigens (HRA). Further studies of the biology and immunochemistry of S. mutans membranes, therefore, appear warranted. Membranes of BHT will be analyzed by crossed immunoelectrophoresis (CIE)-zymogram techniques for adenosine triphosphatase, polynucleotide phosphorylase, aminopeptidase and proteases. Physical properties of enzymes will be determined by nitrocellulose blotting of SDS-PAGE and isoelectric focusing gels followed by zymogram development. Penicillin-binding proteins will be studied in membranes from cells at various growth stages using 14C-benzylpenicillin-binding. The permease, Enzyme II, of the PEP-dependent phosphotransferase system (PTS) will be detected using radioiodinated soluble PTS components as probes to develop blots of two-dimensional gels. Studies of induced vs non-induced cells and PTS-mutants will allow identifications of specific permeases. HRA's in detergent extracts of membranes will be routinely identified by immunoblotting. Purification of membrane-bound or cell-free HRA will be by immunoaffinity chromatography employing monoclonal antibodies (MAb's). Hybridomas will be selected on their abilities to produce MAb's reactive with S. mutans and human heart. Purified HRA's will be chemically analyzed for comparison with wall-localized HRA's. Also, purified antigens will be studied as adherence factors by reacting with heart tissue followed by indirect immunofluorescence and indirect RIA. The further occurrence of membrane HRA's similar to that of BHT will be investigated by immunoblot analyses of membranes from other S. mutans serotypes, other viridans streptococci, and fresh isolates of S. pyogenes from rheumatics. Mab's to BHT HRA will be used in this survey. Finally, the Mab's will be used to develop immunoblots of human heart tissue extracts to confirm the tissue-localization of antigens with identical determinants. These will be compared with immunoblots developed with anti-S. pyogenes serum.